(a) Field of the Invention
The invention relates to a novel method for producing human natural interferon-xcex1 using ex vivo expanded cord blood hematopoietic cells.
(b) Description of Prior Art
Interferons (IFNs) are a class of cytokines with pleiotropic biological activities (Pfeffer, L. M. et al., Cancer Res, 58(12): p. 2489-99, 1998). Originally described as potent anti-viral agents, IFNs are now known to also have anti-proliferative and immunomodulatory activities. IFNs are classified as Type I and Type II depending on their structure and stability in acid medium. Type I IFNs are subclassified by homology of amino acid sequence and type of producing cells as IFN-xcex1 (leukocytes), IFN-xcex2 (fibroblasts) and IFN-xcfx89 (leukocytes). Type II IFN is acid-labile and comprises only IFN-xcex3 produced by activated T cells and NK cells. In opposition to IFN-xcex2 and IFN-xcex3, IFN-xcex1 is molecularly heterogeneous and comprises at least 13 proteins coded by more than 14 genes (Pfeffer, L. M., Semin Oncol, 24(3 Suppl 9): p. S9-63-S9-69, 1997). The similarity between the IFN-xcex1 proteins is between 78% and 95% at the protein level and 79/166 amino acids are conserved in the family. Furthermore allelic forms of IFN-xcex1 genes with variations in 1 to 4 amino acids have been described thus greatly increasing the number of potential IFN-xcex1 proteins (Nyman, T. A., et al., Biochem J, 329(Pt 2): p. 295-302, 1998). The exact reason for the existence of so many IFN-xcex1 species remains unclear since all species exhibit the same biological activities. However the specific activities of each species in different biological assays vary greatly by a factor up to 1000-fold and this observation could be related in some cases to differences in affinity of the various IFN-xcex1 species for the receptor (IFN-xcex1R) expressed on various cells (Pfeffer, L. M., Semin Oncol, 24(3 Suppl 9): p. S9-63-S9-69, 1997). It remains to be seen if the mixture of IFN-xcex1 species varies according to the producing cells or the inducing agent.
In the 1970s, the anti-proliferative activity of Type I IFNs attracted much interest for potential use in cancer treatment. At that time, the available IFN-xcex1 was natural (nIFN-xcex1) and produced mainly by overnight culture of pooled human blood leukocytes after infection with Sendai virus following protocols developed by the group of Cantell in Helsinki (Cantell, K., S., et al., Methods Enzymol, 78(Pt): p. 29-38, 1981). This preparation of nIFN-xcex1 has been recently shown to contain at least 9 of the known IFN-xcex1 species (Nyman, T. A., et al., Biochem J, 329(Pt 2): p. 295-302, 1998). The molecular cloning of the first IFN-xcex1 cDNA (species 2a) in 1979 shifted the interest to recombinant molecules (rIFN-xcex1) produced in bacteria because of the possibility of large supply which was difficult to achieve with the leukocyte-derived nIFN-xcex1. In 1987, the first rIFN-xcex1 was approved by the FDA for use in the treatment of hairy cell leukemia. This rIFN-xcex12a molecule was followed shortly after by a rIFN-xcex12b species (Baron, S., et al., Journal of American Medicine Association, 266(10), p. 1375-1383, 1991). Today the rIFN-xcex1s are widely used in the treatment of more than 10 malignancies and virologic diseases including the widespread hepatitis B and C infections (Pfeffer, L. M. et al., Cancer Res, 58(12): p. 2489-99, 1998). However therapeutic use rIFN-xcex1 results in clinical improvement in only a fraction of the patient populations. For example, the rIFN-xcex1 treatment of Hepatitis C-infected patients is highly effective only in 30% of the cases. Significant side effects of rIFN-xcex1 injection are routinely observed and may prevent the long-term treatment necessary to eradicate the virus. Also a significant proportion of IFN-xcex1-treated patients (10-20%) develop antibody inhibitors which may interfere with the therapeutic effect or prevent continuous treatment. These limitations and side effects and the fact that the two available rIFN-xcex1 species (2a and 2b) may not be the most effective IFN-xcex1 species in some diseases have renewed the interest in the nIFN-xcex1 preparations. Indeed less side effects and frequency of antibody inhibitors formation have been observed in some small scale clinical trials. Also the switch from rIFN-xcex1 to nIFN-xcex1 could permit to prolong the treatment of patients which have developed a resistance to rIFN-xcex1. With the same objectives, a synthetic rIFN-xcex1 termed rIFN-xcex1-con1 has been designed in vitro by assigning at each position in the primary sequence, the amino acid most frequently observed in several IFN-xcex1 species. The rIFN-xcex1-con1 is also tested in clinical trials.
The nIFN-xcex1 is currently produced by overnight culture of Sendai virus-infected human leukocytes isolated from the buffy coats prepared from several hundred blood donations (Cantell, K., S., et al., Methods Enzymol, 78(Pt): p. 29-38, 1981). This procedure has several limitations. On one hand, it requires tight logistics with the blood bank since production of nIFN-xcex1 must be done with fresh cells and initiated within 24 hours of blood collection. In this regard, the increasing use of pre-storage leukodepletion by filtration to reduce contamination of red blood cells and platelets by leukocytes will further increase the difficulties in recovering leukocytes for nIFN-xcex1 production. On the other hand, lots of nIFN-xcex1 must be prepared from pools of leukocytes prepared from thousands of blood donations. Although the nIFN-xcex1 can be highly purified and subjected to viral inactivation procedures, there are concerns about possible contamination of the final product with untested or unknown infectious agents.
Much work has been done to characterize the IFN-xcex1 producing cells present in the peripheral blood. Early results showed that the major IFN-xcex1 producing cells constituted only a minor portion of blood leukocytes (Feldman, S. B., et al., Virology, 204(1): p. 1-7, 1994; and Brandt, E. R., et al., Br J Haematol, 86(4): p. 717-725, 1994). Subsequent work showed that these cells possessed markers characteristic of immature monocyte/dendritic cells (Eloranta, M. L., et al., Scand J Immunol, 46(3): p. 235-241, 1997; and Svensson, H., et al., Scand J Immunol, 44(2): p. 164-172 1996). Recently a major IFN-xcex1-producing cell in blood was isolated and shown to have markers characteristics of lymphoid dendritic cells (DC) precursors (CD4+CDllcxe2x88x92) (Siegal, F. P., et al., Science, 284(5421): p. 1835-1837, 1999). Dendritic cells are terminally differentiated lymphoid and myeloid cells that have important immunomodulatory roles in antigen presentation and cytokine secretion. Mature DCs are constantly produced from both lymphoid and myeloid precursors (Hart, D. N., Blood, 90 (9): p. 3245-3287, 1997). One strategy to increase the nIFN-xcex1 productivity of blood leukocytes would be to expand the IFN-xcex1-producing cells in vitro prior to IFN-xcex1 induction with Sendai virus. Culture conditions (GMxe2x88x92CSF+IL4) that permits to expand the blood DCs have been described but the expansion factor remained limited (10-20xc3x97) (Romani, N., et al., J Exp Med, 180(1): p. 83-93, 1994). Also the presence of cytotoxic T lymphocytes would prevent the pooling of the leukocytes from different donors for the culture expansion phase. However hematopoietic stem cells (HSCs) can now be expanded in vitro for several weeks (Piacibello, W., et al., Blood, 89(8): p. 2644-2653, 1997; Traycoff, C. M., et al., Exp Hematol, 26 (1): p. 53-62, 1998; and Ziegler, B. L. and L. Kanz, Curr Opin Hematol, 5(6): p. 434-440, 1998). In these cultures, the HSCs proliferate and differentiate autonomously into progenitors of the various blood cell lineages. But in most instances, differentiation of HSCs is not complete and does not proceed to the mature blood cell stage in these cultures (Ziegler, B. L. and L. Kanz, Curr Opin Hematol, 5(6): p. 434-440, 1998).
It would be highly desirable to be provided with a method for producing human natural interferon-xcex1 on a large scale.
One aim of the present invention is to provide a method for producing human natural interferon-xcex1 on a large scale using ex vivo hematopoietic stem cells such as cord blood hematopoietic cells.
In accordance with the present invention there is provided a process for producing ex vivo natural interferon xcex1 (nIFN-xcex1) from cultured hematopoietic stem cells (HSCs) infected with Sendai Virus.
Still in accordance with the present invention, there is provided a method for producing ex vivo natural interferon xcex1 (nIFN-xcex1). The method comprises the steps of:
a) infecting hematopoietic stem cells (HSCs) in culture medium with Sendai Virus; and
b) culturing the HSCs of step a) for a time sufficient for the HSCs to produce nIFN-xcex1.
The method of the present invention may further comprise before step a) the step of expanding under suitable conditions the HSCs in culture.
In another embodiment of the present invention, the method may further comprise before step a) and after the step of expanding, a step of pooling expanded HSCs obtained from the step of expanding.
The method of the present invention may further comprise before step a) and after the step of expanding if any, the step of priming the HSCs for nIFN-xcex1 secretion by incubating the HSCs in IFN-xcex1.
The method of the present invention may further comprise after step b), a step of collecting from the culture medium the natural nIFN-xcex1 obtained from step b).
Preferably, the HSCs are from cord blood. Also, the HSCs are preferably enriched in CD34+.
In accordance with the present invention, there is also provided a natural interferon-xcex1 produced by the method described above.